HIV-1 Gag Compact form Stabilized by Intramolecular Interactions is Crucial for Infectious Particle Production.

HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm.

Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO).

The human cellular protein NoL12 is a specific partner of the HIV-1 nucleocapsid protein NCp7.

The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7.

Upstream of N-Ras (Unr/CSDE1) Interacts with NCp7 and Gag, Modulating HIV-1 IRES-Mediated Translation Initiation.

The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC) as a mature protein or as a domain of the Gag precursor plays important roles in the early and late phases of the infection. To better understand its roles, we searched for new cellular partners and identified the RNA-binding protein Unr/CSDE1, Upstream of N-ras, whose interaction with Gag and NCp7 was confirmed by co-immunoprecipitation and FRET-FLIM. Unr interaction with Gag was found to be RNA-dependent and mediated by its NC domain.

Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage.

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins.

pH-Dependent absorption spectrum of oxyluciferin analogues in the active site of firefly luciferase.

In the quest for the identification of the light emitter(s) responsible for the firefly bioluminescence, the study of oxyluciferin analogues with controlled chemical and electronic structures is of particular importance. In this article, we report the results of our experimental and computational investigation of the pH-dependent absorption spectra characterizing three analogues bound into the luciferase cavity, together with adenosine-monophosphate (AMP). While the analogue microscopic pKa values do not differ much from their reference values, it turns out that the AMP protonation state is analogue-dependent and never doubly-deprotonated.

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations.

How HIV-1 Gag Manipulates Its Host Cell Proteins: A Focus on Interactors of the Nucleocapsid Domain.

The human immunodeficiency virus (HIV-1) polyprotein Gag (Group-specific antigen) plays a central role in controlling the late phase of the viral lifecycle. Considered to be only a scaffolding protein for a long time, the structural protein Gag plays determinate and specific roles in HIV-1 replication. Indeed, via its different domains, Gag orchestrates the specific encapsidation of the genomic RNA, drives the formation of the viral particle by its auto-assembly (multimerization), binds multiple viral proteins, and interacts with a large number of cellular proteins that are needed for its functions from its translation location to the plasma membrane, where newly formed virions are released.

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study.

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway.

Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane.

The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions.

5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein.

The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties.

A Class of Potent Inhibitors of the HIV-1 Nucleocapsid Protein Based on Aminopyrrolic Scaffolds.

The HIV-1 nucleocapsid protein 7 (NC) is a potential target for effective antiretroviral therapy due to its central role in virus replication, mainly linked to nucleic acid (NA) chaperone activity, and low susceptibility to drug resistance. By screening a compounds library, we identified the aminopyrrolic compound CN14_17, a known carbohydrate binding agent, that inhibits the NC chaperone activity in the low micromolar range. Different from most of available NC inhibitors, CN14_17 fully prevents the NC-induced annealing of complementary NA sequences.

(Thia)calixarenephosphonic Acids as Potent Inhibitors of the Nucleic Acid Chaperone Activity of the HIV-1 Nucleocapsid Protein with a New Binding Mode and Multitarget Antiviral Activity.

The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity.

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection.

Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations.

ReAsH/tetracystein-based correlative light-electron microscopy for HIV-1 imaging during the early stages of infection.

Visualization of viruses in the host cell during the course of infection by correlative light-electron microscopy (CLEM) requires a specific labelling of the viral structures in order to recognize the nanometric viral cores in the intracellular environment. For Human immunodeficiency virus type 1 (HIV-1), the labelling approaches developed for fluorescence microscopy are generally not suited for transmission electron microscopy (TEM), so that imaging of HIV-1 particles in infected cells by CLEM is not straightforward. Herein, we adapt the labeling approach with a tetracystein tag (TC) and a biarsenical resorufin-based label (ReAsH) for monitoring the HIV-1 particles during the early stages of HIV-1 infection by CLEM.

Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains.

HIV/AIDS is still one of the leading causes of death worldwide. Current drugs that target the canonical steps of the HIV-1 life cycle are efficient in blocking viral replication but are unable to eradicate HIV-1 from infected patients. Moreover, drug resistance (DR) is often associated with the clinical use of these molecules, thus raising the need for novel drug candidates as well as novel putative drug targets.

Discovery and Structure-Based Optimization of 2-Ureidothiophene-3-carboxylic Acids as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors.

We are concerned with the development of novel anti-infectives with dual antibacterial and antiretroviral activities for MRSA/HIV-1 co-infection. To achieve this goal, we exploited for the first time the mechanistic function similarity between the bacterial RNA polymerase (RNAP) "switch region" and the viral non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Starting from our previously discovered RNAP inhibitors, we managed to develop potent RT inhibitors effective against several resistant HIV-1 strains with maintained or enhanced RNAP inhibitory properties following a structure-based design approach.

Investigating the cellular distribution and interactions of HIV-1 nucleocapsid protein by quantitative fluorescence microscopy.

The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA.

Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila.

Background: In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. Recently, in Drosophila, we reported that odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) is related to odor duration.

Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity.

We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation).

Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors.

Genetically encoded probes have become powerful tools for non-invasive monitoring of ions, distributions of proteins and the migration and formation of cellular components. We describe the functional expression of two molecular probes for non-invasive fluorescent monitoring of intracellular Cl ([Cl]i) and the functioning of glycine receptor (GlyR) channels. The first probe is a recently developed cyan fluorescent protein-yellow fluorescent protein-based construct, termed Cl-Sensor, with relatively high sensitivity to Cl (Kapp approximately 30 mM).

Prenatal activation of microglia induces delayed impairment of glutamatergic synaptic function.

Background: Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established.